Chromatographic separation of target molecules is of great commercial interest in the chemical and biotechnological fields, such as the large-scale production of novel biological drugs and diagnostic reagents. Furthermore, the purification of proteins has recently become of great significance due to advances in the field of proteomics, wherein the function of proteins expressed by the human genome is studied. Besides proteins, nucleic acids such as plasmid DNA and particles such as virus particles also need to be purified by chromatographic means e.g. in vaccine production and for gene therapy purposes.
In general, proteins are produced in cell culture, where they are either located intracellularly or secreted into the surrounding culture media. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, growth factors, etc. Separation and purification of a desired protein from the complex mixture of nutrients and cellular by-products, to a level sufficient for therapeutic usage, poses a formidable challenge.
Porous polysulphone and cellulosic membranes are widely used for filtering and separating chemical and biological mixtures (cf. EP0483143). These membranes include ultra- and microfiltration membranes, in which the filtration process is based on a hydrostatic pressure differential. Ultra-filtration membranes are characterized by pore sizes which enable them to retain macromolecules having a molecular weight ranging between 500 and 1,000,000 Daltons.
Microfiltration membranes exhibit permselective pores ranging in diameter between 0.01 and 10 microns (μm), as measured by bubble point testing. For adsorptive separations, where membranes are substituted with ligands to bind the target species, microfiltration membranes are typically used. In order to avoid any pore filtration effects it is preferred to use membranes with a pore size >0.1 μm or even >0.5 μm, i.e. well above the size of the largest intended target species and high enough to give a high flow rate. Larger pore sizes give even better flow rates, but with conventional membranes the adsorption capacities will then be too low, due to the lower available surface area.
Despite their widespread usage, cellulosic membranes suffer a number of disadvantages, including susceptibility to attack by strong acids and bases, and by cellulase enzymes. Sensitivity to bases is characterized initially by shrinkage and swelling, ultimately leading to decomposition of the membrane. High temperatures promote chemical disintegration and shrinkage while low temperatures, especially in connection with substantial concentrations of alkali, promote swelling and bursting. The pore structure of the membrane can easily be destroyed resulting in a dramatic decrease in the flow rate through the membrane.
From the experience of the textile industry, it has long been known that better characteristics may be imparted to cellulosic fibers by cross-linking (cf. Kirk-Othmer's Encyclopedia of Chemical Technology, Vol. 22, pp 770-790 (3rd Ed. 1983)). Such cross-linking is particularly desirable in order to improve the physical strength and chemical resistivity of the cellulosic membranes. Furthermore, where chemical derivatization of the membranes is desirable, for example in order to couple protein binding ligands to the hydroxyl groups of the cellulose polymers, base sensitivity is particularly important.
Accordingly, it is an object of the present invention to provide porous cross-linked charged cellulosic polymeric membranes that have a high adsorption capacity for target molecules, such as proteins, nucleic acids and viruses, when substituted with charged ligands. It is a further object of the invention to provide cross-linked cellulosic polymeric membranes in a process that does not adversely affect either their high flux/flow rates nor their minimal protein adsorption and flexibility, and to impart to the membranes an increased resistance to bases in order to allow further chemical modification. It is a further object of the invention to provide membranes prepared by the aforementioned processes. A further object of the invention is to provide methods for separating target molecules from other components in a solution using said membranes based upon the binding affinities of the target molecules.